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HEMATOPOIESIS
From the Hematopoiesis Department, American Red
Cross Holland Laboratory, Rockville, MD; the Research Institute
of Molecular Pathology, Vienna, Austria; the Division of Experimental
Hematology and the Biochemistry Department, St Jude Children's
Research Hospital, Memphis, TN; and the Howard Hughes Medical
Institute, Chevy Chase, MD.
Signal transducers and activators of transcription (STATs) are
intracellular mediators of cytokine receptor signals. Because many
early-acting growth factors have been implicated in STAT5 activation,
this study sought to investigate whether STAT5 may be a transcriptional
regulator of hematopoietic stem cell (HSC) long-term repopulating
activity. To test this possibility, bone marrow (BM) and fetal liver
(FL) cells from mice containing homozygous deletions of both STAT5a and
STAT5b genes (STAT5ab The functional roles of janus kinases (JAKs) and
signal transducers and activators of transcription (STATs) have been
characterized in both hematopoietic and nonhematopoietic
tissues.1 The JAK/STAT signaling axis allows a diverse set
of extracellular signals to result in modification of gene expression
patterns in the appropriate target cells. This process is initiated
when, on cytokine binding, JAKs come into close proximity and become
activated by autophosphorylation on tyrosine residues. Activated JAKs
then phosphoryate among many other substrates, the STAT proteins. STATs
form homodimers or heterodimers via SH2 domain phosphotyrosine
interactions and translocate to the nucleus where they bind to
transcriptional elements on DNA.2,3 Activated STAT
proteins regulate their target genes often with tissue specificity. In
the hematopoietic system, JAK/STAT signaling has been extensively
described in a variety of lymphoid and myeloid cell types. Four
mammalian JAKs and 7 STAT proteins have been identified and
characterized by generation of knockout mice.4 These
studies have revealed a large diversity in the role of STATs in
hematopoiesis with some STATs showing phenotypes primarily in
restricted cytokine pathways (eg, interferons, interleukin [IL]-4,
IL-12, and IL-13) that are primarily involved in immune responses
(STAT1, STAT2, STAT4, and STAT6). In contrast, the complex phenotypes
of STAT3- and STAT5-deficient mice demonstrate a broad cytokine
activation and function profile for many individual cell lineages.
STAT5a and STAT5b are 2 very homologous transcription factors with
variability between these 2 proteins being primarily in the
transactivation domain. Mice deficient in either STAT5a or STAT5b have
been generated and characterized. The STAT5a knockout mouse is
characterized by defects in responses to granulocyte-macrophage colony-stimulating factor (GM-CSF)5 and mammary gland
development.6 The STAT5b knockout mouse is characterized
by defects in growth hormone signaling and expression of
male-characteristic liver gene expression patterns.7 To
study the effects of STAT5 on hematopoiesis and also to eliminate any
compensating function between the 2 STAT isoforms, homozygous mutant
mice lacking both STAT5a and STAT5b were generated. These female mice
were infertile, but heterozygous mice could breed normally and give
viable STAT5ab Many of the growth factors reported to activate STAT514-21
have been shown to provide a survival signal for primitive
hematopoietic cells during ex vivo culture. For example, IL-3, IL-6,
stem cell factor (SCF),22,23 Flt3 ligand,24
and thrombopoietin (TPO)25-28 have been shown to stimulate
primitive hematopoietic cells to proliferate ex vivo when present at
the optimal concentrations and combinations. Tyrosine phosphorylation
of STAT5 in megakaryocytes and platelets after TPO treatment has been
well characterized.29-32 Therefore, given the activity of
TPO on multipotential cells and the stem cell-repopulating defect in
mice lacking the receptor for TPO (c-mpl),33-36 we were
interested in studying the role of STAT5 in the stem cell compartment.
Furthermore, TPO is known to be synergistic with other early-acting
cytokines such as SCF and Flt3 ligand.27 For applications
such as ex vivo retroviral gene transfer using hematopoietic cytokines,
STAT5 activation may also be required for the synergistic effects of
growth factor stimulation. The requirement for STAT5 as a common
signaling intermediate in primitive levels of hematopoiesis had not
been previously studied. Therefore, we have tested the hypothesis that
a block in signal transduction pathways involving STAT5 could result in
decreased stem cell activity in hematopoietic cells from
STAT5ab Mice and genotyping
Mouse peripheral blood hematology
Bone marrow/fetal liver cell collection and spleen colony-forming unit assays Bone marrow (BM) was harvested from both hind limbs (tibias and femurs) of either STAT5ab / or littermate wild-type
mice. BM cells were flushed into phosphate-buffered saline containing
2% fetal bovine serum (Hyclone, Logan UT) and counted using a
hemacytometer. Embryonic day 14.5 fetuses were collected after the
pregnant females were humanely killed, and the liver cells were
removed with a pair of sterile microforceps, dispersed with a 21-gauge
needle, and resuspended in phosphate-buffered saline containing 2%
fetal bovine serum. A small fraction (1:20) of the fetal liver (FL)
cells was then used the same day for STAT5 genotyping by PCR as
described above. The total number of BM cells obtained from each mouse
was used for calculation of the absolute number of spleen
colony-forming units (CFU-Ss) in the BM based on the frequency. CFU-S
assays were performed by using BM cells pooled from either 2 wild-type
or STAT5ab/ mice. CFU-Ss were harvested 12 days after
injection of standard doses of 5 × 104 or
1 × 105 cells via the lateral tail vein into recipient
mice irradiated with 900 rads of gamma radiation 2 to 4 hours earlier
(137Cs source; Mark I-68A model irradiator, J. L. Shepherd and Associates, San Fernando, CA). Manual scoring of CFU-S
colonies was aided by the use of a StereoZoom 6 Plus dissecting
microscope (Leica Microsystems, Buffalo, NY). Preliminary
characterization of CFU-S numbers in STAT5ab/ mice was
performed with 1 × 104, 5 × 104,
1 × 105, and 1 × 106 cells to find the
linear range. The numbers of CFU-Ss per spleen for the data in Figure
1B ranged from 4 to 10 using the
1 × 105 cell dose. A dose dependence on colony formation
was observed for all 3 experiments.
Antibody/fluorescent dye staining and flow cytometry BM cells were flushed from both hind limbs of STAT5ab/ and wild-type mice. BM cells were stained with
a cocktail of antibodies to phycoerythrin (PE)-conjugated lineage
markers that included Ly-6G (Gr-1), CD11b (Mac-1), CD45R/B220, CD4
(L3T4), CD8 (Ly-2), NK1.1 (NKR-P1B and NKR-P1C), and Ter119/Ly-76. The
cells were also stained with antibodies to fluorescein isothiocyanate
(FITC)-conjugated Ly-6A/E (Sca-1) and biotin-conjugated CD117 (c-kit).
The biotinylated c-kit antibody was detected by using a secondary
streptavidin-PE Cy5 conjugate. FL cells were collected as described
above and were stained with the same lineage cocktail except that CD11b (Mac-1) was not included.37 All antibodies for these
studies were obtained from PharMingen (San Diego, CA). For flow
cytometric analysis, cells were stained with 5 µg/mL Hoechst 33342 as
previously reported38 before antibody staining on ice.
Cells were then analyzed by using a FACSVantage SE flow cytometer (BD
Biosciences, San Jose, CA) equipped with the Enterprise IIC laser,
providing both 488 nm and UV (351-364 nm) excitation. The primary beam
was tuned to 488 nm at 100 mW for light scatter triggering, and the second beam was tuned to UV at 30 mW for Hoechst 33342 excitation. Fluorescence emission from bound Hoechst 33342 dye was measured at 2 wavelengths, blue fluorescence emission detected with a 424/44 bandpass
filter and red fluorescence emission with a 675 longpass filter.
Separation of the 2 signals was achieved with a 640-nm longpass
dichroic filter. Linear signals from both red and blue fluorescence
channels were collected and used to produce typical histograms for
identification of side-population (SP) cells. Small debris was
excluded from the analysis by electronic gating of the forward versus
orthogonal light scatter data. The SP cell gate was defined according
to normal C57Bl/6 BM and was similar to that reported38,39
The PE-conjugated lineage antibodies described also including Thy1.2
antibody were used for staining peripheral blood leukocyte populations
in mice that received transplants. The percentage of Ly-5.2 donor
engraftment in Ly-5.1 recipient mice was quantitated by using a
FITC-conjugated CD45.2 (anti-Ly-5.2) antibody in combination with the
PE-conjugated lineage antibodies.
Competitive repopulation assays BM cells were freshly harvested from both hind limbs of STAT5ab/ and littermate wild type mice. FL cells were
freshly harvested from embryonic day 14.5 fetuses and were genotyped
the same day by PCR. Cells were then mixed thoroughly at either 1:1 or
4:1 donor equivalent ratios with HW80 or Ly-5.1 BM or FL cells
collected at the same time. The cell mixtures were then injected via
the lateral tail vein into lethally irradiated (1100 rads) recipient mice. The minimum cell dose injected was 1.5 to 2 × 106
cells for each competed BM graft and 3 to 5 × 105 cells
for each FL graft. The average mixing ratio was
4.7 × 106 wild-type BM cells versus
2.1 × 106 STAT5ab/ BM cells, because the
BM cellularity for the STAT5ab/ mice was reduced. For
FL experiments, the average competed cells for wild- type and
STAT5ab/ was not different because FL cellularity was
not reduced (see "Results" section). Beginning at 8 weeks after
transplantation, mice were bled from the retroorbital venous plexus.
Hemoglobin patterns were analyzed from packed peripheral red blood
cells by electrophoresis on cellulose acetate gels. To calculate the relative proportions of single and diffuse donor hemoglobin in peripheral blood from reconstituted mice,40 the hemoglobin
gels were digitized by using a ScanJet IIcx/T scanner (Hewlett Packard, Palo Alto, CA). Data files were quantitated by densitometry using ImageQuant software (Molecular Dynamics). For experiments in which donor grafts were competed by using the Ly-5.1/Ly-5.2 system, mice were
analyzed by flow cytometry as described above.
Secondary BM transplantations BM was harvested from primary recipients at times up to 6 months after transplantation and injected via the lateral tail vein into lethally irradiated secondary recipients (1100 rads). Secondary transplanted mice received at least 5 × 106 BM cells each. Hemoglobin electrophoresis patterns were monitored in secondary recipients after reconstitution (4 months). For some analyses, the recipient mice were Ly-5.1 and engraftment was determined by FACS.Southern blot analyses Genomic DNA was prepared from peripheral blood, BM, and spleen cells from mice that received transplants by digestion with 0.6 mg/mL proteinase K in 50 mM Tris pH 8.0, 1% sodium dodecyl sulfate, 100 mM NaCl, and 10 mM EDTA pH 8.0. DNA was then extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 2.5 volumes ice-cold ethanol and one-tenth volume sodium acetate. DNA (5-10 µg) was digested overnight with EcoRI and separated on a 0.8% agarose gel by electrophoresis. Gels were blotted overnight onto Hybond N+ nylon membrane (Amersham, Arlington Heights, IL), UV cross-linked, and hybridized with a [32P]-labeled fragment of the mouse -globin
intervening sequence 2. Blots were washed at a final stringency of
0.5 × SSC/0.5% sodium dodecyl sulfate at 65°C and exposed
overnight, and autoradiographic images were obtained by using a
Molecular Dynamics Storm phosphorimager and x-ray film (Eastman Kodak,
Rochester, NY). [32P]-(deoxycytidine triphosphate) dCTP
was obtained from Amersham.
STAT5ab mice 8 generations onto the C57Bl/6 background.
Heterozygote mice were then crossed to yield homozygous double-
knockout mice for analysis. The survival of homozygous mutant mice on
the C57Bl/6 background was very low, with 4-week survival of
approximately 5% of all mice genotyped. We have used the C57Bl/6
STAT5ab/ mice to further characterize the hematopoietic
defects relative to normal littermate wild-type mice and to determine
whether reduced long-term repopulating activity results from loss of
STAT5. The peripheral blood hematology of the backcrossed mice is shown
in Table 1. The peripheral white blood
cell counts were decreased because of a reduced percentage of
lymphocytes, and the hematocrits were near normal, except in aged mice
(> 3 months old) that developed splenomegaly and were not analyzed. A
mild increase in the absolute neutrophil count and absolute monocyte
count was also noted. This result is similar to our previous
observations in JAK3/ mice, suggesting a compensatory
mechanism for a decreased absolute lymphocyte count. Not shown in Table
1, we also determined the absolute red blood cell and platelet counts
by using the hematology analyzer for mice that did not receive
transplants. The red blood cell numbers were 67% of normal in the
STAT5ab/ mice (wild type
[8.2 ± 1.0 × 106/µL] versus
STAT5ab/ [5.5 ± 0.8 × 106/µL]
n = 6, P = .004). The platelet numbers were 40% of
normal in the STAT5ab/ mice (wild type
[1288 ± 271 × 103/µL] versus
STAT5ab/ [516 ± 107 × 103/µL] n = 6, P < .001).
For our studies, BM was collected from young mice (4-6 weeks) to avoid
age-related disease and death. Note that, because of the young age of
these mice, the BM cellularity from the wild type mice was slightly
lower than that typical of older mice. BM was harvested from both hind
limbs and, relative to littermate wild type mice,
STAT5ab STAT5ab (KLS)
(Figure 2A). The absolute number of these
cells was not different between wild-type and STAT5ab/
mice in either the BM or FL. The absolute number of KLS cells in the FL
(7218 ± 2234, n = 4) relative to normal littermate FL (9746 ± 2407, n = 5) was unchanged (P = .15).
Accordingly, the percentage of FL KLS cells (wild type
[0.25 ± 0.05%, n = 5] versus STAT5ab/
[0.24 ± 0.07%, n = 4]) and the FL cellularity (wild type
[2.1 ± 0.74 × 106, n = 11] versus
STAT5ab/ [2.1 ± 0.75 × 106,
n = 9]) did not differ between wild-type and
STAT5ab/ mice (P = .81 and 1.0, respectively). The adult BM from these mice also showed a normal
absolute number of KLS cells despite a reduction in total BM
cellularity (Figure 1). The absolute number of hind limb KLS cells was
quantitated at 23 010 ± 19 950 in STAT5ab/ mice
(n = 5) relative to 16 754 ± 12 566 for wild type mice
(n = 6), which was not significantly different
(P = .54). For the FL samples, a second method was used
that was based on Hoechst 33342 dye efflux (Figure 2B). The SP cells
reported after dual-emission wavelength analysis has been shown to be
enriched for repopulating cells, but they also contain lineage-positive
cells such as natural killer cells and erythroid progenitors
(Ter119+).41,42 We found that 60% to 70% of
BM KLS cells fall within the SP cell gate; therefore, significant
overlap occurs between these 2 parameters. The
c-kit+lin SP and
Sca-1+lin SP cell populations were
quantitated from both wild-type and STAT5ab/ FL cells.
No significant difference between wild-type and
STAT5ab/ FL SP cell percentage was found by using
either positive selection for c-kit or Sca-1. The
c-kit+lin SP number was 15 120 ± 4366 in
wild-type mice (n = 4) and 17 640 ± 8250 in
STAT5ab/ mice (n = 4), which was not significant
(P = .61). The Sca-1+lin SP
number was much lower and included only 420 ± 148 cells in wild-type
mice (n = 5) and 210 ± 75 cells in STAT5ab/ mice
(n = 4, but the difference between wild-type and
STAT5ab/ mice was significant (P = .04).
However, because the Sca-1+lin SP fraction
was much lower than the c-kit+lin SP
fraction, the total numbers of STAT5ab/ SP cells were
not reduced.
Further studies demonstrated that reconstitution of hematopoiesis could
be achieved after direct transplantation of 5 × 106
STAT5ab
BM from STAT5ab / mice with those from littermate wild-type mice
to determine whether differences in relative stem cell activity were
evident. For these experiments, BM cells were harvested from either
STAT5ab/ or littermate wild-type mice (C57Bl/6
background; Hbs) and competed at a 1:1 ratio with cells
from HW80 mice (Hbd). Mixing based on ratio was important
because total BM cellularity does not necessarily correlate with
absolute stem cell content. Beginning as early as 7 weeks after
transplantation and continued monthly until 6 months before
transplantation, mice from BM competitive repopulation experiment no. 1 were analyzed for the relative levels of donor engraftment (Figure
5A). The Hbs band was always
absent even at the earliest time points for the
STAT5ab/ grafts in all experiments. BM from wild-type
mice (nos. 8-10) competed effectively with the competitor marrow,
resulting in mice with the expected chimerism (38% ± 6%). In
contrast, BM from STAT5ab/ mice was completely
outcompeted by the competitor marrow, indicating a greatly reduced
long-term repopulating activity (mice nos. 1-7). At the time the mice
from the competitive repopulation experiment (mice nos. 1, 2, 4, 9, and
10) were humanely killed, secondary transplanted mice were generated
and followed for 4 months to continue analysis (Figure 5B). The average
percentage of Hbs in 7 secondary transplanted mice was not
significantly changed from the average found in the primary
transplanted mice after wild-type competitive repopulation
(32% ± 10%, n = 7, P = .37). As in the primary
transplantation, no reconstitution was seen in secondary recipients
from the STAT5ab/ competitive repopulation. The defects
in secondary reconstitution rule out a phenotype restricted to
short-lived progenitors. Two additional competitive repopulation
experiments analyzed at 4 and 3 months after transplantation showed
identical results for the STAT5ab/ BM graft (experiment
no. 2, n = 5; experiment no. 3, n = 8) and the wild-type graft
(experiment no. 2, 54% ± 3%, n = 3; experiment no. 3, 56%,
n = 2). The average percentage of chimerism in the 1:1 mixed
wild-type grafts for the 3 separate experiments was 49% ± 10%.
To confirm the hemoglobin reconstitution analysis results by Southern
blot for the single and diffuse alleles, primary transplanted mice were
killed at 6 months, and genomic DNA was prepared from leukocytes
obtained from peripheral blood, BM, and spleen (SP) of mice nos. 2, 4, 9, and 10. For mouse no. 1 only peripheral blood was obtained for
analysis. Southern blot analysis of the DNA from mice in each
experimental group was then performed (Figure 6). The Southern blots were probed with a
fragment of the
FL cells harvested from STAT5ab / mice, we characterized a more
primitive stage of hematopoietic cell development in the competitive
repopulation assay. FL cells were mixed at 1:1 donor ratios, and,
because the FL cellularity was unchanged, the cell doses competed were
identical. When equal donor equivalents from pools of 2 wild-type or
STAT5ab/ FL were mixed with HW80 mouse FL cells, the
reconstitution from the STAT5ab/ graft was completely
outcompeted at all times measured up to 20 weeks after transplantation
(Figure 7A). This result was identical to
that obtained with adult BM, indicating that autoreactive T cells,
which are absent at this developmental stage, are not responsible for
the dysfunction in hematopoietic reconstitution on BM or FL transplantation. A second experiment was performed in the hemoglobin system with identical results (STAT5ab/ n = 5, wild
type n = 3, data not shown). A third experiment was also performed
using the CD45 marker after mixing of FL cells from 2 separate donor
mice with Ly-5.1 FL cells at either a 1:1 or 4:1 ratio. A
representative example of both wild-type and STAT5ab/
competitions is shown (Figure 7B) from a total of 5 recipients of 1:1
mixed cells and 3 recipients of 4:1 mixed cells for both the wild-type
and STAT5ab/ groups. This approach complements the
hemoglobin electrophoresis results and provides quantitative
multilineage characterization of the repopulating defects. We have
mixed STAT5ab/ (Ly-5.2) or wild-type E14.5 FL cells
harvested from the same pregnant female (Ly-5.2) at donor equivalent
ratios of 1:1 or 4:1 versus wild-type E14.5 FL cells from a Ly-5.1
pregnant female. Multilineage engraftment was determined by costaining
for Gr-1, Mac-1, B220, CD4, or Ter119 markers and for Ly-5.2 at 10 and
17 weeks after transplantation, with no changes observed between the 2 time points. Wild-type livers competed with values consistent with the
input 50% (28%-59% range) or 80% (61%-93% range) mixing ratio.
Even with the 4:1 mixing ratio, the STAT5ab/ graft only
contributed from undetectable to 3.7%. The levels of engraftment of
CD4+ T cells were always undetectable for the
STAT5ab/ graft and in rank order were B220+
B cells (1.3%-1.6%), Ter119+ erythroid progenitors
(1.6%-1.7%), Gr-1+ myeloid cells (3.3%-4.6%), and
Mac-1+ myeloid cells (3.7%-4.7%). The net overall
repopulating deficiency was thus greatest in T cells (because
undetectable) and could be quantitated at 25- to 28-fold for
granulocytes (Gr-1) and macrophage (Mac-1), 45-fold for erythroid
progenitors (Ter119), and 68-fold for B lymphocytes. This result
formally demonstrates the lymphomyeloid nature, the severity, and the
generalizability of the repopulating defect in hematopoietic cells from
STAT5ab/ mice.
Many of the growth factors that have characterized biologic
activities on primitive hematopoietic cells have been shown to induce
phosphorylation of STAT5 via the JAKs. Although this indirect evidence
suggested that STAT5 activation might be important for cytokine
stimulation of HSCs, no functional studies using STAT5-deficient mice
had been performed. In addition, other signal transduction pathways
apart from the JAK-STAT pathway could serve redundant functions, making
the role of JAKs and STATs in HSCs uncertain. For example, both TPO and
SCF can activate other downstream molecules such as SHC, Raf-1/MAP
kinase, and phosphatidylinositol-3' kinase43-49 in various
cell types. The requirement for any of these pathways in the HSC has
not been directly studied. To address the role of STAT5 signaling in
the earliest stages of hematopoietic differentiation, we have initiated
studies using a STAT5ab As previously reported, we found that STAT5ab |
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Abstract
Introduction
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